ptsG基因敲除对肺炎克雷伯氏菌CCR效应的缓解及发酵产2,3-BD的影响

doi:10.11924/j.issn.1000-6850.casb2023-0075

中国农学通报 ›› 2024, Vol. 40 ›› Issue (3): 95-102.doi: 10.11924/j.issn.1000-6850.casb2023-0075

ptsG基因敲除对肺炎克雷伯氏菌CCR效应的缓解及发酵产2,3-BD的影响

毛亮阳(), 李娜, 葛菁萍()

黑龙江大学，生命科学学院，农业微生物技术教育部工程研究中心，黑龙江省区植物基因与生物发酵重点实验室，黑龙江省普通高校微生物重点实验室，哈尔滨 150080

收稿日期:2023-01-29 修回日期:2023-05-15 出版日期:2024-01-17 发布日期:2024-01-17
通讯作者:
葛菁萍，女，1972年出生，黑龙江齐齐哈尔人，教授，博士，研究方向：微生物资源挖掘与利用。通信地址：150080 黑龙江省哈尔滨市南岗区学府路74号黑龙江大学224信箱，Tel：0451-86609016，E-mail：gejingping@126.com。
作者简介:
毛亮阳，男，2000年出生，海南儋州人，硕士，研究方向：微生物资源挖掘与利用。通信地址：150080 黑龙江省哈尔滨市南岗区学府路74号黑龙江大学生命科学学院，Tel：0451-86609016，E-mail：1432218771@qq.com。
基金资助:
黑龙江省自然科学基金重点项目“乙酸代谢与副干酪乳杆菌群体感应互作探究及对细菌素产生的影响”(ZD2020C008); 黑龙江省生态环境厅2022年度生态环境保护科研项目“人工重组细菌菌群对黑土磷障碍的解控机制”(HST2022TR004); 2023年黑龙江省高校基本科研业务费“群体感应介导下重组根圈细菌菌群对土壤磷养分循环的调控机制”

ptsG Knockout: The Impact on CCR Effect of Klebsiella pneumoniae and Its Fermentation Production of 2,3-BD

MAO Liangyang(), LI Na, GE Jingping()

Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education/Heilongjiang Provincial Key Laboratory of Plant Genetic Engineering and Biological Fermentation Engineering for Cold Region/Key Laboratory of Microbiology, College of Heilongjiang Province/School of Life Sciences, Heilongjiang University, Harbin 150080

Received:2023-01-29 Revised:2023-05-15 Published-:2024-01-17 Online:2024-01-17

摘要/Abstract

摘要：

旨在解决Klebsiella pneumoniae在利用混合碳源发酵生产2,3-丁二醇（2,3-butanediol，简称2,3-BD）时会产生碳分解代谢抑制(Carbon catabolite repression，CCR)效应，导致生产效率下降的问题。本研究以Cm抗性基因为标记，采用λRed同源重组技术成功构建ptsG基因缺失菌株K. pneumoniae HD79-N。此外，在利用葡萄糖与木糖混合碳源（葡萄糖:木糖=2:1）发酵的结果显示，K. pneumoniae HD79-N菌株由于敲除ptsG基因显著减弱了CCR效应，使其能够同步利用葡萄糖与木糖生产2,3-BD且最终产量达9.81±0.38 g/L。同时，K. pneumoniae HD79-N[0.23±0.01 g/(L·h)]菌株木糖利用率也比K. pneumoniae HD79[0.15±0.00 g/(L·h)]菌株提高了57.82%。本研究结果为缓解ptsG基因对K. pneumoniae菌株的CCR作用及提升2,3-BD的产量提供技术参考。

关键词: 2,3-丁二醇, 基因敲除, ptsG基因, 同源重组, 肺炎克雷伯氏菌

Abstract:

The objective is to address the issue of Carbon Catabolite Repression (CCR) in Klebsiella pneumoniae during mixed carbon source fermentation for 2,3-butanediol (2,3-BD) production, which leads to a decrease in production efficiency. In this study, a Cm resistance gene was used as a marker, and the ptsG gene deletion strain of K. pneumoniae HD79-N was successfully constructed using the λRed homologous recombination technique. Furthermore, the fermentation results using a mixed carbon source of glucose and xylose (glucose:xylose=2:1) demonstrated that the K. pneumoniae HD79-N strain, with the ptsG gene deletion, significantly alleviated CCR, enabling simultaneous utilization of glucose and xylose for 2,3-BD production with a final yield of 9.81±0.38 g/L. Moreover, the xylose utilization rate of K. pneumoniae HD79-N strain [0.23±0.01 g/(L·h)] was also increased by 57.82% compared to that of K. pneumoniae HD79 strain [0.15±0.00 g/(L·h)]. The findings of this study provide technical insights into alleviating the CCR effect caused by the ptsG gene in K. pneumoniae strains and enhancing the production of 2,3-BD.

Key words: 2,3-butanediol, gene knockout, ptsG gene, homologous recombination, Klebsiella pneumonia

毛亮阳, 李娜, 葛菁萍. ptsG基因敲除对肺炎克雷伯氏菌CCR效应的缓解及发酵产2,3-BD的影响[J]. 中国农学通报, 2024, 40(3): 95-102.

MAO Liangyang, LI Na, GE Jingping. ptsG Knockout: The Impact on CCR Effect of Klebsiella pneumoniae and Its Fermentation Production of 2,3-BD[J]. Chinese Agricultural Science Bulletin, 2024, 40(3): 95-102.

图/表 11

参考文献 27

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序号	名称	菌株用途	菌株特点	菌株来源
1	K. pneumoniae HD79	出发菌株	野生型	黑龙江大学微生物重点实验室保存
2	E. coli DH5α	克隆和转化	ΔlacU169, Rˉ, Mˉ, Amp^r	黑龙江大学微生物重点实验室保存
3	K. pneumoniae HD79-N	用于本探究	Cm^r, ΔptsG, Cm^r	自行构建

序号	名称	菌株用途	菌株特点	菌株来源
1	K. pneumoniae HD79	出发菌株	野生型	黑龙江大学微生物重点实验室保存
2	E. coli DH5α	克隆和转化	ΔlacU169, Rˉ, Mˉ, Amp^r	黑龙江大学微生物重点实验室保存
3	K. pneumoniae HD79-N	用于本探究	Cm^r, ΔptsG, Cm^r	自行构建

序号	名称	质粒特点	质粒用途	质粒来源
1	pMD18-T	Amp^r, lacZ, 2692 bp	克隆载体	大连宝生物有限公司
2	pKD46	Amp^r, Exo, Beta, Gam, 6329 bp	同源重组	北京鼎国生物科技有限公司
3	pLysS	Cm^r, T7 lysozyme, 4886 bp	克隆cm基因	北京鼎国生物科技有限公司

序号	名称	质粒特点	质粒用途	质粒来源
1	pMD18-T	Amp^r, lacZ, 2692 bp	克隆载体	大连宝生物有限公司
2	pKD46	Amp^r, Exo, Beta, Gam, 6329 bp	同源重组	北京鼎国生物科技有限公司
3	pLysS	Cm^r, T7 lysozyme, 4886 bp	克隆cm基因	北京鼎国生物科技有限公司

引物名称	引物序列(5’-3’)	用途
ptsG₁-F	CCGTACTGCCTATCGCAGGTATC	克隆ptsG₁上游片段594 bp
ptsG₁-R	gacaccaggCGTTCCGATGTGATGCAGACC	克隆ptsG₁上游片段594 bp
ptsG₂-F	tggtgctacgcGCGATTTGGCACTCTGCTAAAC	克隆ptsG₂下游片段579 bp
ptsG₂-R	GTACGCCAGAACCTGCCACTAC	克隆ptsG₂下游片段579 bp
cm^r-F	GATCATCTGAACGCGGCACGTAAGAGGTTCC	克隆cm^r序列888 bp
cm^r-R	CACGCCATTCCCCGCTTATTATCACTTATTCA	克隆cm^r序列888 bp
pKD46-F	CCCAGCCCTGTGTATAACTCAC	验证转化结果 1387 bp
pKD46-R	CAACTCGGTCGCCGCATACA	验证转化结果 1387 bp
ptsG-F	AGCCAAAGCGAGTAAAGTTCAC	验证重组结果2507 bp
ptsG-R	GCGGACTGTTTTCAGGGTTAT	验证重组结果2507 bp
M13R	GTAAAACGACGGCCAGT	验证重组质粒
M13F	TGTAAAACGACGGCCAGT	验证重组质粒
ptsG-up	CGTGATCCTCTCCTTCATTTG	qRT-PCR检测扩增ptsG
ptsG-down	tAAGCCGCCAGACAGTTTG	qRT-PCR检测扩增ptsG
xylA-up	CGGGCGAAGTTATTGCTG	qRT-PCR检测扩增xylA
xylA-down	GGCTTTCCGTCATTACAACC	qRT-PCR检测扩增xylA
xylB-up	CCGCCGATAAGCGTGAT	qRT-PCR检测扩增xylB
xylB-down	GGTGTGGTTCCTGCCCTAT	qRT-PCR检测扩增xylB
xylE-up	TTCCGCACCGAACATCC	qRT-PCR检测扩增xylE
xylE-down	GCCCGCTTTACATTGCC	qRT-PCR检测扩增xylE
xylF-up	ACCTTCCTGCTTGGCTTCT	qRT-PCR检测扩增xylF
xylF-down	aCTGGCAGAAAGACCGTGAT	qRT-PCR检测扩增xylF
xylG-up	GCAGTTCGGATGAAATGACA	qRT-PCR检测扩增xylG
xylG-down	tCCAGCAAAAGGCGATTC	qRT-PCR检测扩增xylG
xylH-up	ACCCACCATCCGTTCCA	qRT-PCR检测扩增xylH
xylH-down	GCCATCGTCGTCATTATGC	qRT-PCR检测扩增xylH
16S rDNA-up	TGTGTAGCCCTGGTCGTAAG	qRT-PCR检测扩增内参基因
16S rDNA-down	CGGACATCCACAGAACTTAGC	qRT-PCR检测扩增内参基因

ptsG基因敲除对肺炎克雷伯氏菌CCR效应的缓解及发酵产2,3-BD的影响

ptsG Knockout: The Impact on CCR Effect of Klebsiella pneumoniae and Its Fermentation Production of 2,3-BD

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